1. Field of the Invention
The present invention provides inhibitors of oligosaccharyl transferase (OT), methods for obtaining Glc.sub.3 Man.sub.9 (GlcNAc).sub.2 -P-P-Dol, methods for obtaining glycopeptides and novel classes of antifungals,' antitumors and antivirals.
2. Discussion of the Background
The first committed step in the biosynthesis of all N-linked glycoproteins is catalyzed by the enzyme oligosaccharyl transferase and involves the transfer of a complex carbohydrate from a dolichol-linked pyrophosphate donor to the carboxamide side-chain of an asparagine residue (FIG. 1). Protein glycosylation is essential for the structure and function of many proteins and is involved in the control of many diverse biological processes (Paulson, Trends in Biol. Sci., 1989, 14, 272; Sadler, In Biology of Carbohydrates, 2nd Ed., Ginsburg & Robbins, Ed., John Wiley & Sons: New York, 1984, Vol. 2, pg. 87). For example, protein glycosylation has been found to be crucial for the development, growth and proper function of complex organisms, while, the aberrant glycosylation of proteins has been associated with diseased and transformed cells.
Despite the centrality of this biochemical transformation there are no synthetic or naturally occurring inhibitors that specifically target this enzyme-catalyzed reaction. In lieu of an inhibitor for OT, researchers have relied on the use of inhibitors of the dolichol phosphate cycle (FIG. 2).
For the synthesis of N-linked glycoproteins, the lipid linked oligosaccharide intermediate is Glc.sub.3 Man.sub.9 (GlcNAc).sub.2 -P-P-Dolichol. This precursor unit is assembled by a series of reactions onto a lipid carrier, dolichol phosphate (reviewed by Parodi & Leoir, Biochem. Biophys. Acta, 1979, 550, 1-37; Sharon & Lis, In The Proteins, 3rd Ed., Neurath & Hill, Ed., Academic Press: London, 1975, Vol. 5, pg. 1). Dolichol serves to firmly anchor the carbohydrate close to the lumen side of the ER membrane near to the site of the emerging polypeptide.
The assembly of the oligosaccharide begins with the reversible transfer of N-acetylglucosamine 1-phosphate from UDP-GlcNAc to Dolichol-P (FIG. 2), followed by the irreversible transfer of N-acetylglucosamine, also from UDP-GlcNAc, to the GlcNAc-P-P-Dol to give (GlcNAc).sub.2 -P-P-Dol. The .beta.-linked mannose residues of the pentasaccharide core are transferred directly from GDP-Man to (GlcNAc).sub.2 -P-P-Dol. Attachment of the remaining mannose residues, as well as of the three glucoses, is believed to proceed via the corresponding dolichol phosphate derivatives, formed respectively from GDP-Man and UDP-Glc. The ultimate product of this cycle is Glc.sub.3 Man.sub.9 (GlcNAc).sub.2 -P-P-Dol. The oligosaccharide is transferred en bloc from the lipid carrier to the nascent polypeptide. Thus, the final step of the dolichol cycle results in the transfer of a mannose-rich oligosaccharide chain to a polypeptide.
Tunicamycin, a glucosamine-containing antibiotic isolated from Streptomyces lysosuperificus, inhibits the first step in the dolichol phosphate cycle, the formation of GlcNAc-P-P-Dol (Tkacz & Lampen, Biochem. Biophys. Res. Commun., 1975, 65, 248). Other inhibitors include 2-deoxyglucose, 2-fluoroglucose, and 2-fluoromannose (Schwarz & Heath, Proc. Natl. Acad. Sci. USA, 1980, 77, 3811). These inhibitors compete for available dolichol phosphate and lead to formation of altered intermediates. However, these compounds are not recognized by OT and therefore inhibition of the overall reaction occurs only as dolichol phosphate supplies become exhausted.
Tunicamycin is described in U.S. Pat. Nos. 4,336,333 and 4,330,624. Unfortunately, tunicamycin is not specific for OT.
Further, tunicamycin is an extremely potent compound and must be used with extreme care. Thus, it is desirable to find inhibitors which are both specific for OT and whose toxicity can be controlled.
Inhibitors specific for OT would be useful both in pharmaceutical applications and in bioengineering applications.
Imperiali et al. (JACS, 1992, 114, 7942) describe a weak inhibitor (1 mM) of OT. The present application describes inhibitors of OT which are 10,000 times more potent than those previously described.